ERBB2 (her-2/neu) overexpression is observed in 30% of all breast cancers and is associated with decreased survival time and therapy response. The modest response rates to targeted therapies of patients with ERBB2-positive metastatic disease has prompted the search for new therapeutic targets other than ERBB2. Our studies with a high throughput functional genomics approach have shown that the genes most critical for the survival of ERBB2-positive breast cancer cells are related to the production and storage of fats. Two of these genes, NR1D1 and PBP, map closely to the ERBB2 locus and are commonly found on amplicons that are responsible for ERBB2 overexpression. NR1D1 functions in adipogenisis and circadian clock regulation and has been proposed as a potential link between the two. PBP is a co-activator of PPAR?. Downregulation of NR1D1 or PBP induces apoptosis in ERBB2-positive breast cancer cells but has no effect on normal breast cells or other breast cancer cells. Antagonists to PPAR? have very similar effects. Our studies indicate that these three genes function as part of a pathway that causes a significant increase in the levels of stored fats compared to other normal or cancerous breast cells. This pathway is required specifically for ERBB2-positive breast cancer cell survival. We hypothesize that increased activity of the NR1D1 pathway enables these cells to coordinately regulate de novo fatty acid synthesis such that energy production is maximized and toxic end products minimized. Thus the tight genetic linkage between NR1D1 and ERBB2 that results in co-overexpression of these two gene products genetically programs these cells to depend on fatty acid synthesis for energy production and survival. The work proposed here is a comprehensive analysis of the genetic programs regulated by NR1D1 in concert with PBP and PPAR? in ERBB2-positive breast cancer cells. This will determine how the physiology of these cells differs from normal breast cells and the effects that this genetic program has on tumorigenesis in vivo. Genetic interactions between NR1D1 and ERBB2 will be characterized and potential synergistic effects of NR1D1 and ERBB2 on cell proliferation, apoptosis resistance, Herceptin sensitivity and fat metabolism identified. The effects of the NR1D1 pathway on the metabolism of ERBB2-positive breast cancer cells will be explored to identify potential mechanisms for increased cancer cell survival. Genes whose expression is regulated in breast cancer cells by the NR1D1 pathway will be identified by molecular profiling and tested for relevance to cancer cell survival using RNAi. The importance of NR1D1 overexpression in the genesis of mammary tumors in mice will be determined. Because NR1D1 is required for survival by human breast cancer cells that overexpress ERBB2 and because it is tightly linked to ERBB2, we will test the hypothesis that NR1D1 overexpression accelerates tumor development and enhances metastatic frequency in mouse models of breast cancer. This may lead to an improved mouse model and a better understanding of the genetic, dietary and circadian influences on the etiology of ERBB2-positive breast cancer. PUBLIC HEALTH RELEVANCE: We have identified a gene that when inhibited kills her-2 positive breast cancer cells but has no effect on normal breast cells. The normal function of this gene is to regulate circadian rhythm and fat storage. The study of this gene may provide information on how diet and "the night shift" contribute to breast cancer and may lead to therapies that target this aggressive tumor type.